JP5907483B2 - Method for producing plant transformed so as to promote dedifferentiation or redifferentiation, transformant, and chimeric protein, chimeric gene, DNA, recombinant expression vector and kit used in the method - Google Patents

Description

  The present invention relates to a method for producing a plant modified so as to promote dedifferentiation or redifferentiation, a transformant, and a protein, chimeric protein, gene, chimeric gene, DNA, recombinant expression vector used in the method And relating to the kit.

  Since many flower garden plants are not genetically fixed (purified), when these are used as research materials, products, etc., techniques for growing clones by culture or the like are required. This culture technique is also an important technique used in the process of producing a plant transformed by genetic recombination.

  As one method of culture growth, there is a method through callus growth (dedifferentiation) from tissue pieces such as leaf pieces, and adventitious bud formation (redifferentiation) from there, but the ease of transition to each stage is The efficiency of this transfer is the most important point for increasing the efficiency of culture growth and transformation.

  For example, transformation and adventitious bud formation of Torenia (Torenia fournieri Lind.), Which is one of the flower garden plants, usually takes about 60 days until adventitious buds start to appear. The number of adventitious buds was about 5% of the number, and the efficiency was very poor.

  In addition, the period for obtaining a transformant of a plant body varies depending on the plant species, but for example, it is about 4 months at the shortest for Torenia, about half a year for tobacco, chrysanthemum, etc., about 1 year for roses, carnations, etc. For example, it takes about 1.5 years to 2 years for flowering, and depending on the state of the plant used as the material and the nature of the transgene, there are cases where only a few lines of genetically modified organisms can be obtained.

  For example, in Patent Document 1, dedifferentiation is promoted by introducing a recombinant expression vector containing a gene encoding a transcription factor involved in dedifferentiation into a plant cell and producing the transcription factor in the plant cell. It is described to produce a plant body modified in this way.

JP 2006-325588 A

  The object of the present invention is to produce a plant modified so as to promote dedifferentiation or redifferentiation, a transformant, and a protein, chimeric protein, gene, chimeric gene, DNA, recombinant used in the method It is to provide expression vectors and kits.

  The present invention allows a plant to produce a chimeric protein in which a transcription factor that promotes transcription of a gene that suppresses dedifferentiation or redifferentiation and a functional peptide that converts any transcription factor into a transcriptional repression factor are fused. This is a method for producing a plant that promotes dedifferentiation or redifferentiation of the plant.

In the plant production method, the transcription factor is preferably the following protein (a ) .
(A) protein having the amino acid sequence shown in SEQ ID NO: 1

In the plant production method, the transcription factor promotes transcription of a gene having 90 % or more identity to the amino acid sequence represented by SEQ ID NO: 1 and suppressing dedifferentiation or redifferentiation. A protein having a function is preferable.

In the plant production method, the following gene (c) or (d) is preferably used as the gene encoding the transcription factor.
(C) A gene having the base sequence shown in SEQ ID NO: 2 as an open reading frame region (d) Hybridizing under stringent conditions with a gene containing a base sequence complementary to the gene containing the base sequence shown in SEQ ID NO: 2 And a gene encoding a transcription factor that promotes transcription of a gene that suppresses dedifferentiation or redifferentiation

In the plant production method, the functional peptide is preferably a peptide having an amino acid sequence represented by any of the following formulas (1) to (4).
(1) X1-Leu-Asp-Leu-X2-Leu-X3
(Here, in Formula (1), X1 represents 0 to 10 amino acid residues, X2 represents Asn or Glu, and X3 represents at least 6 amino acid residues.)
(2) Y1-Phe-Asp-Leu-Asn-Y2-Y3
(Here, in Formula (2), Y1 represents 0 to 10 amino acid residues, Y2 represents Phe or Ile, and Y3 represents at least 6 amino acid residues.)
(3) Z1-Asp-Leu-Z2-Leu-Arg-Leu-Z3
(Here, in Formula (3), Z1 represents Leu, Asp-Leu or Leu-Asp-Leu, Z2 represents Glu, Gln or Asp, and Z3 represents 0 to 10 amino acid residues.)
(4) Asp-Leu-Z4-Leu-Arg-Leu
(Here, in formula (4), Z4 represents Glu, Gln or Asp.)

  In the said plant body production method, it is preferable that the said functional peptide is a peptide which has an amino acid sequence shown in either of sequence number 3-19.

In the method for producing a plant body, the functional peptide is preferably the following peptide (e) or (f).
(E) SEQ ID NO: 20 or a peptide having an amino acid sequence shown in 21 (f) for the amino acid sequence shown in SEQ ID NO: 20 or 21 peptides having at least 90% identity

In the method for producing a plant body, the functional peptide is preferably a peptide having an amino acid sequence represented by the following formula (5).
(5) α1-Leu-β1-Leu-γ1-Leu
(In the formula (5), α1 represents Asp, Asn, Glu, Gln, Thr or Ser, β1 represents Asp, Gln, Asn, Arg, Glu, Thr, Ser or His, and γ1 represents , Arg, Gln, Asn, Thr, Ser, His, Lys or Asp.)

In the plant production method, the functional peptide is preferably a peptide having an amino acid sequence represented by any of the following formulas (6) to (8).
(6) α1-Leu-β1-Leu-γ2-Leu
(7) α1-Leu-β2-Leu-Arg-Leu
(8) α2-Leu-β1-Leu-Arg-Leu
(Here, in each formula (6)-(8), α1 represents Asp, Asn, Glu, Gln, Thr or Ser, α2 represents Asn, Glu, Gln, Thr or Ser, and β1 represents Asp, Gln, Asn, Arg, Glu, Thr, Ser or His, β2 represents Asn, Arg, Thr, Ser or His, γ2 represents Gln, Asn, Thr, Ser, His, Lys or Asp (Shown.)

  In the method for producing a plant body, the functional peptide is preferably a peptide having an amino acid sequence represented by any one of SEQ ID NOs: 22 to 37, 133, 59, and 60.

  In the method for producing a plant body, the functional peptide is preferably a peptide having the amino acid sequence represented by SEQ ID NO: 38 or 39.

  The method for producing a plant includes a transformation step of introducing a recombinant expression vector containing a chimeric gene having a gene encoding the transcription factor and a polynucleotide encoding the functional peptide into a plant cell. preferable.

  The plant production method preferably further comprises an expression vector construction step of constructing the recombinant expression vector.

  The present invention is a plant that has been modified by the method for producing a plant so as to promote dedifferentiation or redifferentiation.

  The present invention is a chimeric protein in which a transcription factor that promotes transcription of a gene that suppresses dedifferentiation or redifferentiation and a functional peptide that converts an arbitrary transcription factor into a transcriptional repression factor are fused.

  The present invention relates to a chimeric gene comprising a gene encoding a transcription factor that promotes transcription of a gene that suppresses dedifferentiation or redifferentiation, and a polynucleotide encoding a functional peptide that converts any transcription factor into a transcriptional repression factor It is.

  The present invention is a DNA comprising a portion encoding a transcription factor that promotes transcription of a gene that suppresses dedifferentiation or redifferentiation, and a portion encoding a functional peptide that converts any transcription factor into a transcriptional repression factor .

  The present invention relates to a chimeric gene comprising a gene encoding a transcription factor that promotes transcription of a gene that suppresses dedifferentiation or redifferentiation, and a polynucleotide encoding a functional peptide that converts any transcription factor into a transcriptional repression factor A recombinant expression vector comprising

  The present invention relates to a chimeric gene comprising a gene encoding a transcription factor that promotes transcription of a gene that suppresses dedifferentiation or redifferentiation, and a polynucleotide encoding a functional peptide that converts any transcription factor into a transcriptional repression factor A transformant comprising a recombinant expression vector comprising

  The transformant is preferably a plant.

  The present invention is a kit for producing a plant, which converts a gene encoding a transcription factor that promotes transcription of a gene that suppresses dedifferentiation or redifferentiation, and an arbitrary transcription factor into a transcriptional repression factor. A kit comprising a recombinant expression vector comprising a chimeric gene having a polynucleotide encoding a functional peptide.

  In the present invention, a plant body modified to promote dedifferentiation or redifferentiation can be produced by suppressing the function of a gene that suppresses the dedifferentiation or redifferentiation of plant cells.

It is process drawing which shows the construction method of the vector for construction for constructing the recombinant expression vector used in an Example. FIG. 5 is a process diagram for incorporating a gene encoding the transcription repressor conversion peptide SRDX and the MYB106 gene into the construction vector p35SG used in the Examples. FIG. 3 is a process diagram showing a method for constructing a transformation vector pBCKH. It is a graph which shows the relationship between the days (days) after infection and the number of shoot appearance callus in Example 1 and Comparative Examples 1-3 of this invention.

  Embodiments of the present invention will be described below. This embodiment is an example for carrying out the present invention, and the present invention is not limited to this embodiment.

  The inventors of the present invention have used a method of gene recombination (CRES-T method) that changes the properties of plants by suppressing the functions of the genes of the plants themselves to produce a large number of genes of Arabidopsis thaliana as flower gardening. In the process of introducing into a plant model Torenia and selecting useful traits, it was found that when a specific gene was introduced, more gene recombinants were obtained than those introduced with other genes. By newly identifying a gene that suppresses dedifferentiation or redifferentiation of plant cells and using this gene, a plant body modified so that dedifferentiation or redifferentiation is promoted can be produced. Thereby, the culture | cultivation of a plant body and the efficiency of transformation can be improved.

  For example, adventitious buds are formed in a conventional period of about 2/3 (about 40 days in the case of Torenia using hygromycin as a drug used for selection of transformants). In this case, about twice the conventional adventitious buds can be obtained in about 60 days.

  The method for producing a plant according to the present embodiment has utility in a field having a certain effect by promoting dedifferentiation or redifferentiation. For example, the plant production method according to the present embodiment shortens the period of production of transformants and increases the number of productions, thereby greatly improving the work efficiency in culture growth, transformation, and the like. A transformant can be obtained in a short period of time, for example, about 2/3, and the necessary number of lines can be easily secured. Moreover, labor saving and cost reduction are possible in culturing flower garden plants and the like that need to be vegetatively grown by culturing.

  The plant production method according to the present embodiment is a technique for suppressing the function of a gene that suppresses dedifferentiation or redifferentiation of plant cells, and a transcription factor that promotes transcription of a gene that suppresses dedifferentiation or redifferentiation. And a chimeric protein in which an arbitrary transcription factor is fused with a functional peptide that converts the transcription factor into a transcriptional repressor. Thereby, transcription of a gene that suppresses dedifferentiation or redifferentiation of plant cells is suppressed, and dedifferentiation or redifferentiation of plant cells is induced and promoted.

  Here, dedifferentiation or redifferentiation of plant cells is promoted as follows. That is, the transcription factor-derived DNA binding domain in the chimeric protein binds to a target gene presumed to suppress dedifferentiation or redifferentiation. The transcription factor is converted into a transcription repressing factor, and transcription of the target gene is repressed. As a result, the production of proteins presumed to suppress dedifferentiation or redifferentiation is reduced, and as a result, dedifferentiation or redifferentiation of plant cells can be promoted.

<Chimeric protein>
As described above, the chimeric protein used in the plant production method according to the present embodiment converts a transcription factor that promotes transcription of a gene that suppresses dedifferentiation or redifferentiation, and an arbitrary transcription factor into a transcriptional repression factor. The functional peptide is fused.

  In addition, the chimeric protein used in the plant production method according to the present embodiment acts dominantly on the endogenous gene, so the plant is diploid or diploid, Or even if a function duplication gene exists in a plant, the expression of the gene which suppresses dedifferentiation or redifferentiation can be suppressed. Therefore, the dedifferentiation or redifferentiation of plant cells is promoted for any plant into which genes can be introduced.

[Transcription factor]
The transcription factor that promotes transcription of a gene that suppresses dedifferentiation or redifferentiation is not particularly limited as long as it is a transcription factor that promotes transcription of a gene that suppresses dedifferentiation or redifferentiation of plant cells. Such transcription factors include transcription factors having similar functions that various plants have.

  A typical example of such a transcription factor is, for example, MYB106 protein. The MYB106 protein is a protein having the amino acid sequence shown in SEQ ID NO: 1, and is one of the MYB family proteins of Arabidopsis thaliana. In this embodiment, for example, the MYB106 protein, which is a transcription factor, is converted into a transcription repressing factor by fusing a functional peptide described later to the MYB106 protein.

  The transcription factor used in the present embodiment is not limited to the MYB106 protein having the amino acid sequence shown in SEQ ID NO: 1, and any transcription factor that promotes transcription of a gene that suppresses dedifferentiation or redifferentiation. Good. Specifically, in the amino acid sequence shown in SEQ ID NO: 1, even a protein having an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and / or added has the above function. If it does, it can be used. The range of “one or more” in the above “amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence shown in SEQ ID NO: 1” Although not particularly limited, for example, it means 1 to 20, preferably 1 to 10, more preferably 1 to 7, still more preferably 1 to 5, and particularly preferably 1 to 3.

  The transcription factor is, for example, a protein having a homology of 20% or more, preferably 50% or more, more preferably 60% or more, and further preferably 70% or more with respect to the amino acid sequence shown in SEQ ID NO: 1. In addition, a protein having a function of promoting transcription of a gene that suppresses dedifferentiation or redifferentiation is also included. Here, “homology” is the ratio of the same sequence in the amino acid sequence, and the higher this value, the closer the two are.

  When producing the chimeric protein used in the present embodiment, a known gene recombination technique can be suitably used as described later. Therefore, the gene encoding the transcription factor can also be suitably used in the plant production method according to the present embodiment.

  The gene encoding the transcription factor is not particularly limited as long as it corresponds to the amino acid sequence of the transcription factor based on the genetic code. As a specific example, for example, when MYB106 protein is used as a transcription factor, a gene encoding this MYB106 protein (hereinafter sometimes referred to as “MYB106 gene”) may be mentioned. As a specific example of the MYB106 gene, for example, a polynucleotide containing the base sequence shown in SEQ ID NO: 2 (Accession No. AY519581, AGI code At3g01140) as an open reading frame (ORF) can be mentioned.

Of course, the MYB106 gene or the gene encoding the transcription factor used in this embodiment is not limited to the above example, and is a gene having homology with the base sequence shown in SEQ ID NO: 2. Also good. Specifically, for example, a gene that hybridizes under stringent conditions with a gene having a base sequence complementary to the gene having the base sequence shown in SEQ ID NO: 2 and that encodes the above transcription factor can be mentioned. .

The hybridization, J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2 nd Ed., Can be performed by a conventionally known method such as methods described in Cold Spring Harbor Laboratory (1989). Usually, the higher the temperature and the lower the salt concentration, the higher the stringency (the more difficult it becomes to hybridize).

  The method for obtaining the gene encoding the transcription factor is not particularly limited, and it can be isolated from many plants by a conventionally known method. For example, a primer pair prepared based on a known transcription factor base sequence can be used. By using this primer pair, the above gene can be obtained by a method of performing PCR using plant cDNA or genomic DNA as a template. The gene encoding the transcription factor can also be obtained by chemical synthesis by a conventionally known method.

[Functional peptides]
The functional peptide used in the present embodiment to convert an arbitrary transcription factor into a transcription repressing factor (hereinafter sometimes referred to as “transcription repressing conversion peptide”) is not particularly limited. Any peptide that can suppress transcription of a target gene controlled by the transcription factor by forming a fused chimeric protein may be used. Specifically, for example, transcription repressor conversion peptides found by the present inventors (Japanese Patent Laid-Open Nos. 2001-269177, 2001-269178, 2001-29276, and 2001-292777). No., JP-A-2001-269176, JP-A-2001-269179, WO 03/055903, Ohta, M., Matsui, K., Hiratsu, K., Shinshi, H. and Ohme- Takagi, M., The Plant Cell, Vol. 13, pp. 1959-1968, August, 2001, Hiratsu, K., Ohta, M., Matsui, K., Ohme−Takagi, M., FEBS Letters, 514 ( 2002), pp.351-354, etc.).

  The present inventors have found that a protein obtained by binding an AtERF3 protein, an AtERF4 protein, an AtERF7 protein, or an AtERF8 protein derived from Arabidopsis thaliana, which is one of the Class II ERF gene groups, significantly suppresses gene transcription. I was getting. Thus, an effector plasmid containing a gene encoding each of the above proteins and DNA excised therefrom was constructed and introduced into a plant cell, thereby succeeding in actually suppressing the transcription of the gene (for example, the above-mentioned JP-A-2001-269177, JP-A-2001-269178, JP-A-2001-292776, JP-A-2001-292777). In addition, a gene encoding tobacco ERF3 protein (for example, see JP-A-2001-269176), rice OsERF3 protein (for example, see JP-A-2001-269179), which is one of the Class II ERF gene groups, In addition, when the same test as described above was performed for genes encoding Arabidopsis thaliana ZAT10 and ZAT11, which are one of the gene groups of zinc finger proteins, it was found that gene transcription is suppressed. Furthermore, the present inventors have revealed that these proteins have a common motif including aspartic acid-leucine-asparagine (DLN) in the carboxyl terminal region. As a result of examining the proteins having this common motif, the protein that suppresses gene transcription may be a peptide having a very simple structure, and the peptide having this simple structure can be used as a transcriptional repressor. It has been found that it has a function of converting to.

  Further, the present inventors have found that the Arabidopsis SUPERMAN protein has a motif that does not match the above-mentioned common motif, but has a function of converting an arbitrary transcription factor into a transcription repressor, and a gene encoding the SUPERMAN protein. It has also been found that a chimeric gene in which is bound to a DNA-binding domain of a transcription factor or a gene encoding a transcription factor produces a protein having a strong transcription repressing ability.

  Therefore, as an example of the transcriptional repression converting peptide used in the present embodiment, the Arabidopsis derived AtERF3 protein, the AtERF4 protein, the AtERF7 protein, the AtERF8 protein, the tobacco ERF3 protein, the rice OsERF3 protein, the zinc finger, which are Class II ERF proteins Examples of such proteins include Arabidopsis thaliana ZAT10 protein and ZAT11 protein, the SUPERMAN protein, peptides excised from these, and synthetic peptides having the above functions.

A specific structure of an example of the transcription repressor converting peptide is a peptide having an amino acid sequence represented by any of the following formulas (1) to (4).
(1) X1-Leu-Asp-Leu-X2-Leu-X3
(Here, in Formula (1), X1 represents 0 to 10 amino acid residues, X2 represents Asn or Glu, and X3 represents at least 6 amino acid residues.)
(2) Y1-Phe-Asp-Leu-Asn-Y2-Y3
(Here, in Formula (2), Y1 represents 0 to 10 amino acid residues, Y2 represents Phe or Ile, and Y3 represents at least 6 amino acid residues.)
(3) Z1-Asp-Leu-Z2-Leu-Arg-Leu-Z3
(Here, in Formula (3), Z1 represents Leu, Asp-Leu or Leu-Asp-Leu, Z2 represents Glu, Gln or Asp, and Z3 represents 0 to 10 amino acid residues.)
(4) Asp-Leu-Z4-Leu-Arg-Leu
(Here, in formula (4), Z4 represents Glu, Gln or Asp.)

(Transcriptional repression converting peptide of formula (1))
In the transcriptional repression converting peptide of the above formula (1), the number of amino acid residues represented by X1 may be in the range of 0 to 10. Moreover, the kind of specific amino acid which comprises the amino acid residue represented by X1 is not specifically limited, What kind of thing may be sufficient. That is, in the transcriptional repression converting peptide of the above formula (1), an oligomer containing one arbitrary amino acid or 2 to 10 arbitrary amino acid residues may be added to the N-terminal side, No amino acids need be added.

  The amino acid residue represented by X1 is preferably as short as possible in view of the ease of synthesizing the transcriptional repression converting peptide of formula (1). Specifically, it is preferably 10 or less, and more preferably 5 or less.

  Similarly, in the transcription repressor converting peptide of the above formula (1), the number of amino acid residues represented by X3 may be at least 6. Moreover, the kind of specific amino acid which comprises the amino acid residue represented by X3 is not specifically limited, What kind of thing may be sufficient. That is, in the transcriptional repression converting peptide of the above formula (1), an oligomer containing 6 or more arbitrary amino acid residues may be added to the C-terminal side. If the amino acid residue represented by X3 is at least 6, it can exhibit the above function.

  In the transcriptional repression converting peptide of the above formula (1), specific sequences of pentamers (5mers) containing 5 amino acid residues excluding X1 and X3 are shown in SEQ ID NOs: 40 and 41. The amino acid sequence when X2 is Asn is the amino acid sequence shown in SEQ ID NO: 40, and the amino acid sequence when X2 is Glu is the amino acid sequence shown in SEQ ID NO: 41.

(Transcriptional repression converting peptide of formula (2))
In the transcriptional repression converting peptide of the above formula (2), as in the case of X1 of the transcriptional repression converting peptide of the above formula (1), the number of amino acid residues represented by Y1 is within the range of 0-10. Good. Moreover, the kind of specific amino acid which comprises the amino acid residue represented by Y1 is not specifically limited, What kind of thing may be sufficient. That is, in the transcriptional repression converting peptide of the above formula (2), as in the transcriptional repression converting peptide of the above formula (1), one arbitrary amino acid or 2 to 10 arbitrary amino acid residues are present on the N-terminal side. An oligomer containing a group may be added, or no amino acid may be added.

  The amino acid residue represented by Y1 should be as short as possible in view of the ease of synthesizing the transcriptional repression converting peptide of formula (2). Specifically, it is preferably 10 or less, and more preferably 5 or less.

  Similarly, in the transcription repressor converting peptide of the above formula (2), the number of amino acid residues represented by Y3 may be at least 6 as in the case of X3 of the transcription repressing converting peptide of the above formula (1). . Moreover, the specific kind of amino acid which comprises the amino acid residue represented by Y3 is not specifically limited, What kind of thing may be sufficient. That is, in the transcriptional repression converting peptide of the above formula (2), as in the case of the transcriptional repression converting peptide of the above formula (1), an oligomer containing 6 or more arbitrary amino acid residues is added to the C-terminal side. Just do it. If the amino acid residue represented by Y3 is at least 6, the above function can be exhibited.

  In the transcription repressor converting peptide of the above formula (2), specific sequences of pentamers (5mers) containing 5 amino acid residues excluding Y1 and Y3 are shown in 42 and 43. The amino acid sequence when Y2 is Phe is the amino acid sequence shown in SEQ ID NO: 42, and the amino acid sequence when Y2 is Ile is the amino acid sequence shown in SEQ ID NO: 43. A specific sequence of tetramer (4mer) containing 4 amino acid residues excluding Y2 is shown in SEQ ID NO: 44.

(Transcriptional repression converting peptide of formula (3))
In the transcriptional repression converting peptide of the above formula (3), the amino acid residue represented by Z1 contains Leu within the range of 1 to 3. The case of 1 amino acid is Leu, the case of 2 amino acids is Asp-Leu, and the case of 3 amino acids is Leu-Asp-Leu.

  On the other hand, in the transcriptional repression converting peptide of the above formula (3), the number of amino acid residues represented by the above Z3 is in the range of 0 to 10 like X1 of the transcriptional repression converting peptide of the above formula (1) If it is. Moreover, the kind of specific amino acid which comprises the amino acid residue represented by Z3 is not specifically limited, What kind of thing may be sufficient. That is, in the transcriptional repression converting peptide of the above formula (3), an oligomer containing one arbitrary amino acid or 2 to 10 arbitrary amino acid residues may be added to the C-terminal side, No amino acids need be added.

  The amino acid residue represented by Z3 should be as short as possible in view of the ease of synthesizing the transcriptional repression converting peptide of formula (3). Specifically, it is preferably 10 or less, and more preferably 5 or less. Specific examples of the amino acid residue represented by Z3 include Gly, Gly-Phe-Phe, Gly-Phe-Ala, Gly-Tyr-Tyr, Ala-Ala-Ala and the like. It is not limited.

  In addition, the number of amino acid residues in the entire transcriptional repressor conversion peptide represented by the formula (3) is not particularly limited, but from the viewpoint of ease of synthesis, it may be 20 amino acids or less. preferable.

  In the transcription repressor converting peptide of the above formula (3), the specific sequence of the oligomer containing 7 to 10 amino acid residues excluding Z3 is shown in SEQ ID NOs: 45 to 53. The amino acid sequence when Z1 is Leu and Z2 is Glu, Gln or Asp is the amino acid sequence shown in SEQ ID NO: 45, 46 or 47, respectively, and Z1 is Asp-Leu and Z2 is Glu, Gln or Asp. Is the amino acid sequence shown in SEQ ID NO: 48, 49 or 50, respectively, and the amino acid sequence when Z1 is Leu-Asp-Leu and Z2 is Glu, Gln or Asp is SEQ ID NO: 51, respectively. The amino acid sequence shown in 52 or 53.

(Transcriptional repression converting peptide of formula (4))
The transcriptional repression converting peptide of the above formula (4) is a hexamer (6mer) containing 6 amino acid residues, and specific sequences thereof are shown in SEQ ID NOs: 7, 16, and 54. The amino acid sequence when Z4 is Glu is the amino acid sequence shown in SEQ ID NO: 7, the amino acid sequence when Z4 is Asp is the amino acid sequence shown in SEQ ID NO: 16, and the amino acid sequence when Z4 is Gln. The sequence is the amino acid sequence shown in SEQ ID NO: 54.

  In particular, the transcription repressor converting peptide used in the present embodiment may be a peptide having a minimum sequence such as hexamer represented by the above formula (4). For example, the amino acid sequence shown in SEQ ID NO: 7 corresponds to the 196th to 201st amino acid sequence of Arabidopsis SUPERMEN protein (SUP protein), and as described above, the present inventors have newly found as the above transcription repressor conversion peptide. Is.

(A more specific example of a transcriptional repressible conversion peptide)
As a more specific example of the transcriptional repression converting peptide represented by each formula described above, for example, a peptide having an amino acid sequence represented by any of SEQ ID NOs: 3 to 19 can be mentioned. These oligopeptides have been found by the present inventors to be the above-described transcriptional repressor converting peptides (see, for example, the above-mentioned WO 03/055903 pamphlet).

Furthermore, the other oligopeptide of the following (e) or (f) description is mentioned as another specific example of the said transcription repression conversion peptide.
(E) Peptide having the amino acid sequence shown in SEQ ID NO: 20 or 21 (f) In the amino acid sequence shown in SEQ ID NO: 20 or 21, one or more amino acids are substituted, deleted, inserted, and / or added. Peptides having a defined amino acid sequence

  The peptide comprising the amino acid sequence shown in SEQ ID NO: 20 is a SUP protein. In addition, “one or more amino acid sequences in which one or more amino acids are substituted, deleted, inserted and / or added in any amino acid sequence shown in SEQ ID NO: 20 or 21” above. The range of “individual” is not particularly limited, and means, for example, 1 to 20, preferably 1 to 10, more preferably 1 to 7, further preferably 1 to 5, and particularly preferably 1 to 3 To do.

  Deletion, substitution or addition of the amino acid can be performed by modifying the base sequence encoding the peptide by a technique known in the art. Mutation can be introduced into the nucleotide sequence by a known method such as Kunkel method or Gappend duplex method or a method equivalent thereto, for example, a mutation introduction kit using site-directed mutagenesis (for example, Mutant- Mutations are introduced using K, Mutant-G (both trade names, manufactured by TAKARA)) or the like, or using LA PCR in vitro Mutagenesis series kits (trade name, manufactured by TAKARA).

  The functional peptide is not limited to the peptide having the full-length sequence of the amino acid sequence shown in SEQ ID NO: 20, and may be a peptide having a partial sequence thereof.

  Examples of the peptide having the partial sequence include a peptide having the amino acid sequence shown in SEQ ID NO: 21 (the 175th to 204th amino acid sequence of the SUP protein). The peptide having the partial sequence includes the above formula ( The peptide represented by 3) is mentioned.

(Other examples of transcription repressor conversion peptides)
The present inventors further examined the structure of the above motif and found a new motif containing 6 amino acids. Specifically, this motif is a peptide having an amino acid sequence represented by the following general formula (5). These peptides are also included in the above-mentioned transcription repressor conversion peptide.
(5) α1-Leu-β1-Leu-γ1-Leu
(In the formula (5), α1 represents Asp, Asn, Glu, Gln, Thr or Ser, β1 represents Asp, Gln, Asn, Arg, Glu, Thr, Ser or His, and γ1 represents , Arg, Gln, Asn, Thr, Ser, His, Lys or Asp.)

The peptides represented by the general formula (5) are classified into peptides having the amino acid sequence represented by the following general formula (6), (7), (8) or (9) for convenience.
(6) α1-Leu-β1-Leu-γ2-Leu
(7) α1-Leu-β2-Leu-Arg-Leu
(8) α2-Leu-β1-Leu-Arg-Leu
(9) Asp-Leu-β3-Leu-Arg-Leu
(Here, in each formula (6)-(9), α1 represents Asp, Asn, Glu, Gln, Thr or Ser, α2 represents Asn, Glu, Gln, Thr or Ser, and β1 represents Asp, Gln, Asn, Arg, Glu, Thr, Ser or His, β2 represents Asn, Arg, Thr, Ser or His, β3 represents Glu, Asp or Gln, γ2 represents Gln, Asn , Thr, Ser, His, Lys or Asp.)

  As more specific examples of the transcriptional repression converting peptide having the amino acid sequence represented by the above formulas (5) to (9), SEQ ID NOs: 22, 23, 24, 25, 26, 27, 28, 29, 30, Peptides having an amino acid sequence represented by 31, 32, 33, 34, 35, 36, 37, 133, 59, or 60 can be mentioned. Among these, the peptide of SEQ ID NO: 29, 30, 32, 34, 133, 59 or 60 corresponds to the peptide represented by the general formula (6), and the peptide of SEQ ID NO: 22, 25, 35, 36 or 37 is It corresponds to the peptide represented by the general formula (7), the peptide of SEQ ID NO: 26, 27, 28, 31 or 33 corresponds to the peptide represented by the general formula (8), and the peptide of SEQ ID NO: 23 or 24 is It corresponds to the peptide represented by the general formula (9).

  In addition to the peptides represented by the above formulas (5) to (9), a transcription repressing conversion peptide having the amino acid sequence represented by SEQ ID NO: 38 or 39 can also be used.

(Chimeric protein production method)
The various transcription repression converting peptides described above can be used as transcription repressing factors by being fused with the transcription factors described above to form chimeric proteins. Therefore, in this embodiment, a chimeric protein can be produced by obtaining a chimeric gene with a gene encoding a transcription factor using the polynucleotide encoding the transcription repressing conversion peptide.

  Specifically, a chimeric gene is constructed by linking a polynucleotide encoding the transcription repressing conversion peptide (hereinafter sometimes referred to as “transcription repressing conversion polynucleotide”) and a gene encoding the transcription factor. And then introduced into plant cells. Thereby, a chimeric protein can be produced. A specific method for introducing the chimeric gene into plant cells will be described later.

  The specific base sequence of the transcriptional repression conversion polynucleotide is not particularly limited, and may include a base sequence corresponding to the amino acid sequence of the transcriptional repression conversion peptide based on the genetic code. In addition, if necessary, the transcriptional repression conversion polynucleotide may include a base sequence serving as a linking site for linking with a transcription factor gene. Further, when the amino acid reading frame of the transcription repressor conversion polynucleotide does not match the reading frame of the transcription factor gene, an additional base sequence for matching them may be included.

  Specific examples of the transcription repression conversion polynucleotide include, for example, SEQ ID NOs: 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, Polynucleotide having the base sequence shown in 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 134, 55, 57 Is mentioned. Also, SEQ ID NOs: 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 135, 56, 58 are polynucleotides complementary to the polynucleotides exemplified above, respectively. It is. In addition, other specific examples of the transcription repressor conversion polynucleotide include, for example, the polynucleotides shown in SEQ ID NOs: 95 and 96. These polynucleotides correspond to the amino acid sequences shown in SEQ ID NOs: 3-39, 133, 59, 60 as shown in Table 1 below.

  The chimeric protein used in the present embodiment can be obtained from the above chimeric gene in which a gene encoding a transcription factor and a transcriptional repression conversion polynucleotide are linked. Therefore, the chimeric protein is not particularly limited as long as it contains the site of the transcription factor and the site of the transcription repressor conversion peptide. For example, a polypeptide having a linker function for linking between a transcription factor and a transcriptional repressor converting peptide, and a polypeptide for epitope-labeling a chimeric protein such as His, Myc, Flag, etc. A polypeptide may be included. Furthermore, the chimeric protein may contain a structure other than a polypeptide, such as a sugar chain or an isoprenoid group, as necessary.

<Example of plant body production method>
The method for producing a plant according to the present embodiment is not particularly limited as long as it includes the process of producing the chimeric protein described above in a plant. If the production method of the plant body which concerns on this embodiment is shown by a specific process, it will be mentioned as a production method including processes, such as an expression vector construction process, a transformation process, and a selection process, for example. Among these, in the present embodiment, it is sufficient that at least the transformation step is included. Hereinafter, each step will be specifically described.

(Expression vector construction process)
The expression vector construction step performed in the present embodiment may be a step of constructing a recombinant expression vector containing the gene encoding the transcription factor described above and the transcription repression-converting conversion polynucleotide described above. It is not limited. The recombinant expression vector may further comprise a promoter.

  Various vectors known in the art can be used as a base vector for the recombinant expression vector. For example, a plasmid, phage, cosmid or the like can be used, and can be appropriately selected according to the plant cell to be introduced and the introduction method. Specific examples include pBR322, pBR325, pUC19, pUC119, pBluescript, pBluescriptSK, pBI vectors, and the like. In particular, when the method of introducing a vector into a plant is a method using Agrobacterium, it is preferable to use a pBI binary vector. Specific examples of the pBI binary vector include pBIG, pBIN19, pBI101, pBI121, pBI221, and the like.

  The said promoter will not be specifically limited if it is a promoter which can express a gene in a plant body, A well-known promoter can be used suitably. Examples of such promoters include cauliflower mosaic virus 35S promoter (CaMV35S), actin promoter, nopaline synthase promoter, tobacco PR1a gene promoter, tomato ribulose 1,5-diphosphate carboxylase oxidase small subunit promoter. Etc. Among these, cauliflower mosaic virus 35S promoter or actin promoter can be used more preferably. When these promoters are used, the obtained recombinant expression vector can strongly express an arbitrary gene when introduced into a plant cell. The promoter is more preferably a promoter capable of specifically expressing a gene during dedifferentiation or redifferentiation. By using such a promoter, the gene encoding the chimeric protein is expressed only at the time of dedifferentiation or redifferentiation, and promotes the dedifferentiation or redifferentiation of the plant body without affecting other tissues. Is possible.

  The promoter may be linked to a chimeric gene in which a gene encoding a transcription factor and a transcriptional repression-converting polynucleotide are linked, and may be introduced into a vector. The structure is not particularly limited.

  The recombinant expression vector may further contain other DNA segments in addition to the promoter and the chimeric gene. The other DNA segment is not particularly limited, and examples thereof include a terminator, a selection marker, an enhancer, and a base sequence for improving translation efficiency. The recombinant expression vector may further have a T-DNA region. The T-DNA region can increase the efficiency of gene transfer particularly when Agrobacterium is used to introduce the recombinant expression vector into a plant body.

  The terminator is not particularly limited as long as it has a function as a transcription termination site, and may be a known one. For example, specifically, the transcription termination region of the nopaline synthase gene (Nos terminator), the transcription termination region of the cauliflower mosaic virus 35S (CaMV35S terminator) and the like can be preferably used. Of these, the Nos terminator can be more preferably used.

  In the above transformation vector, by placing the terminator at an appropriate position, after being introduced into a plant cell, an unnecessarily long transcript is synthesized, or a strong promoter reduces the copy number of the plasmid. Such a phenomenon can be prevented from occurring.

  As the selection marker, for example, a drug resistance gene can be used. Specific examples of such drug resistance genes include drug resistance genes for hygromycin, bleomycin, kanamycin, gentamicin, chloramphenicol and the like. Thereby, the transformed plant body can be easily selected by selecting the plant body growing in the medium containing the antibiotic.

  Examples of the base sequence for improving the translation efficiency include an omega sequence derived from tobacco mosaic virus. By placing this omega sequence in the untranslated region (5'UTR) of the promoter, the translation efficiency of the chimeric gene can be increased. Thus, various DNA segments can be included in the transformation vector according to the purpose.

  The method for constructing the recombinant expression vector is not particularly limited, and the promoter, the gene encoding the transcription factor, the transcription repressor conversion polynucleotide, and, if necessary, the mother vector appropriately selected. What is necessary is just to introduce | transduce said other DNA segment so that it may become a predetermined order. For example, a chimeric gene is constructed by linking a gene encoding a transcription factor and a transcriptional repression-converting polynucleotide, and then linking this chimeric gene and a promoter (such as a terminator if necessary) to form an expression cassette. Build it and introduce it into the vector.

  In the construction of the chimeric gene and the expression cassette, for example, it is possible to define the order of the DNA segments by setting the cleavage sites of each DNA segment as complementary protruding ends and reacting with ligation enzymes. . When the terminator is included in the expression cassette, the promoter, the chimeric gene, and the terminator may be in order from the upstream. Further, the reagents for constructing the recombinant expression vector, that is, the types of restriction enzymes and ligation enzymes are not particularly limited, and commercially available ones may be appropriately selected and used.

  Moreover, the propagation method (production method) of the recombinant expression vector is not particularly limited, and a conventionally known method can be used. In general, Escherichia coli may be used as a host and propagated in the E. coli. At this time, a preferred E. coli type may be selected according to the type of vector.

(Transformation process)
The transformation step performed in the present embodiment is not limited as long as the recombinant expression vector described above is introduced into a plant cell to produce the chimeric protein described above.

  The method (transformation method) for introducing the recombinant expression vector into the plant cell is not particularly limited, and any conventionally known method appropriate for the plant cell can be used. Specifically, for example, a method using Agrobacterium or a method of directly introducing into plant cells can be used. Examples of methods using Agrobacterium include Aida R, Shibata M (1995) Agrobacterium-mediated transformation of torenia (Torenia fournieri). Breed Sci 45: 71-74; Aida R, Kishimoto, Tanaka Y, Shibata M (2000 ) Modification of flower color in torenia (Torenia fournieri Lind) by genetic transformation. Plant Sci 153: 33-42 can be used.

  As a method for introducing a recombinant expression vector directly into a plant cell, for example, a microinjection method, an electroporation method (electroporation method), a polyethylene glycol method, a particle gun method, a protoplast fusion method, a calcium phosphate method, or the like may be used. it can.

  Examples of plant cells into which the recombinant expression vector is introduced include cells of each tissue, callus, suspension culture cells, etc. in plant organs such as flowers, leaves and roots.

  Here, in the method for producing a plant according to the present embodiment, the recombinant expression vector may be appropriately constructed according to the type of plant to be produced. A replacement expression vector may be constructed in advance and introduced into plant cells. That is, the recombinant expression vector construction process described above may or may not be included.

(Other processes, other methods)
In the plant production method according to the present embodiment, it is sufficient if the transformation step is included, and the recombinant expression vector construction step may be further included, but further other steps are included. May be. Specifically, the selection process etc. which select an appropriate transformant from the plant body after transformation are mentioned.

  The method for selecting a plant into which the target gene has been introduced is not particularly limited, and it may be selected based on the expression status of the introduced gene in the plant body. For example, a method of simultaneously introducing a “marker gene” in addition to the target gene can be mentioned. When a drug resistance gene is used as the marker gene, it may be selected by putting an antibiotic or the like in the medium. When an enzyme gene (such as GUS gene) that produces a pigment is used as the marker gene, it may be selected by adding a substrate to the medium. When a fluorescent protein (GFP or the like) that emits fluorescence as a marker gene is used, the fluorescence emitted by the cells may be confirmed and selected by observation with a fluorescence microscope.

  In the method for producing a plant according to this embodiment, since the chimeric gene is introduced into the plant, offspring can be obtained from the plant by sexual reproduction or asexual reproduction. It is also possible to obtain plant cells and propagation materials such as seeds, fruits, strains, callus, tubers, cut ears, lumps and the like from the plant and its descendants, and mass-produce the plant based on these. . Therefore, the production method for a plant according to the present embodiment may include a breeding process (mass production process) for breeding the selected plant.

  In addition, the plant body in this embodiment includes at least one of a grown plant individual, a plant cell, a plant tissue, a callus, and a seed. In other words, in the present embodiment, any plant that can finally grow up to a plant individual is regarded as a plant body. The plant cells include various forms of plant cells. Such plant cells include, for example, suspension culture cells, protoplasts, leaf sections and the like. Plants can be obtained by growing and differentiating these plant cells. In addition, the reproduction | regeneration of the plant body from a plant cell can be performed using a conventionally well-known method according to the kind of plant cell. Therefore, in the method for producing a plant according to the present embodiment, a regeneration step for regenerating the plant from the plant cell may be included.

  Moreover, the production method of the plant body which concerns on this embodiment is not limited to the method of transforming with a recombinant expression vector, You may use another method. Specifically, for example, the chimeric protein itself may be administered to a plant body. In this case, the chimeric protein may be administered to the young plant so that dedifferentiation or redifferentiation can be promoted at the site of the plant finally used. Also, the administration method of the chimeric protein is not particularly limited, and various known methods may be used.

(Plant obtained by this embodiment, its usefulness, and its use)
The method for producing a plant according to the present embodiment is based on expressing a gene encoding the chimeric protein in the plant. A transcription factor-derived DNA binding domain in the chimeric protein binds to a target gene presumed to be involved in promotion of dedifferentiation or redifferentiation. The transcription factor is converted to a transcription repressor, and transcription of the target gene is repressed. Thereby, dedifferentiation or redifferentiation can be promoted. Therefore, this embodiment also includes a plant obtained by the above-described method for producing a plant.

(Specific example of a plant according to the present embodiment)
Here, the specific kind of the plant body in which dedifferentiation or redifferentiation according to the present embodiment is promoted is not particularly limited. For example, a plant whose usefulness is enhanced by promoting dedifferentiation or redifferentiation. Can be mentioned. Such a plant may be an angiosperm or a gymnosperm. As a gymnosperm, for example, a cedar family, a pine family, a cypress family, a asteraceae plant, and the like are cited. Further, the angiosperm may be a monocotyledonous plant or a dicotyledonous plant. Examples of the dicotyledonous plants include plants such as Brassicaceae such as Arabidopsis thaliana and camellia. In addition, examples of monocotyledonous plants include plants such as rice, corn, wheat, etc.

  Moreover, the plant body in which dedifferentiation or redifferentiation according to the present embodiment is promoted may be a plant using fruits and seeds as a product, or a horticultural plant using flowers and plants themselves as products. Specific examples of such plants include rapeseed, potato, spinach, soybean, cabbage, lettuce, tomato, cauliflower, peas, turnip, bean jam, radish, broccoli, melon, orange, watermelon, leek, burdock, etc. There are various edible plants or horticultural plants such as roses, chrysanthemums, hydrangea and carnations.

  The method for producing a plant according to the present embodiment is particularly suitably applied to culture growth and transformation of a genetically modified plant such as a genetically modified flower. Even when a culture of a genetically modified plant is propagated, a transformant can be obtained in a shorter period of time than before, and the necessary number of lines can be easily secured, thereby saving labor and cost. Examples of genetically modified plants include genetically modified flowers such as Torenia (Torenia fournieri Lind.), Chrysanthemum, rose, carnation, rice, soybean, wheat, and the like. By applying the method for producing a plant according to this embodiment, development for producing a more useful genetically modified plant can be performed while reducing time, cost, and labor.

  Torenia is an annual plant of the Azalea pepper family native to Indochina. Torenia can be grown with a small plant height (for example, flowering at a minimum of about 7 cm), can be easily maintained and propagated by cuttings, has a short period of time until it blooms, and has high humidity like a plant box regardless of day length. This embodiment has features such as a diploid (2n = 18) that is normally flowered even under cultivation conditions, a small genome size (171 Mbp = almost equivalent to Arabidopsis thaliana), a genetic recombination technique has been established, and the like. The plant production method according to the above is suitably applied.

(Specific example of transformant according to this embodiment)
Here, according to the present embodiment, a polynucleotide encoding a gene encoding a transcription factor that promotes transcription of a gene that suppresses dedifferentiation or redifferentiation, and a functional peptide that converts any transcription factor into a transcription suppression factor The specific type of transformant containing a recombinant expression vector containing a chimeric gene having the above is not particularly limited. For example, in addition to the transformed plant body, Agrobacterium, E. coli, yeast And viruses.

(An example of utilization of the production method of the plant body concerning this embodiment)
The field of use and the method of using the plant production method according to the present embodiment are not particularly limited, but as an example, a kit for performing the plant production method according to the present embodiment, that is, removal of the plant body. Examples include differentiation or regeneration differentiation kits.

  Specific examples of this plant dedifferentiation or redifferentiation promotion kit include a gene encoding a transcription factor that promotes transcription of a gene that suppresses dedifferentiation or redifferentiation, and an arbitrary transcription factor converted into a transcriptional repression factor. It only needs to contain at least a recombinant expression vector containing a chimeric gene having a polynucleotide encoding a functional peptide. More preferably, it contains a reagent group for introducing the recombinant expression vector into plant cells. Examples of the reagent group include enzymes and buffers according to the type of transformation. In addition, if necessary, experimental materials such as microcentrifuge tubes may be attached.

(Method for producing plant body in which dedifferentiation or redifferentiation is promoted)
The present inventors have clarified that MYB106 protein and similar transcription factors conserved in various plants are transcription factors that promote transcription of genes that suppress dedifferentiation or redifferentiation, and completed the present invention. I came to let you. Therefore, a method for producing a plant body in which the promotion of dedifferentiation or redifferentiation is controlled using a gene encoding such a transcription factor is also included in this embodiment.

That is, the method for producing a plant body in which the promotion of dedifferentiation or redifferentiation according to this embodiment uses a gene encoding a protein described in the following (a) or (b).
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 1,
(B) Transcription of a gene that includes the amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence shown in SEQ ID NO: 1 and suppresses dedifferentiation or redifferentiation Protein with the function of promoting

Or the production method of the plant body by which promotion of dedifferentiation or redifferentiation which concerns on this embodiment uses the gene of the following (c) or (d) description.
(C) a gene having the base sequence shown in SEQ ID NO: 2 as an open reading frame region,
(D) Transcription that promotes transcription of a gene that hybridizes under stringent conditions with a gene containing a base sequence complementary to the gene containing the base sequence shown in SEQ ID NO: 2 and suppresses dedifferentiation or redifferentiation Gene encoding factor

  This plant production method can be achieved by suppressing or over-expressing the gene encoding the transcription factor that suppresses dedifferentiation or redifferentiation. Examples of the method for suppressing the expression of the gene include an antisense method, a gene targeting method, an RNAi method, a co-suppression method, and a gene disruption tagging method. Examples of a method for overexpressing the gene include a method of constructing a vector containing an appropriate promoter and the gene arranged downstream thereof and introducing the vector into a plant.

  Hereinafter, although an example and a comparative example are given and the present invention is explained more concretely in detail, the present invention is not limited to the following examples.

[Example 1]
In this example, a polyenzyme encoding a 12 amino acid peptide LDLDLELLRLGFA (SRDX) (SEQ ID NO: 19), which is one of transcription repressor conversion peptides, between the cauliflower mosaic virus 35S promoter and the transcription termination region of the nopaline synthase gene. Torenia was transformed by constructing a recombinant expression vector incorporating a polynucleotide having a nucleotide linked downstream of the MYB106 gene and introducing it into Torenia using the Agrobacterium method.

<Construction of vector for transformation vector construction>
As shown in FIG. 1, p35SG, which is a vector construction vector for transformation, was constructed as shown in the following steps (1) to (4).

  (1) The regions of attL1 and attL2 on the pENTR vector manufactured by Invitrogen Corporation are designated as primers attL1-F (SEQ ID NO: 138), attL1-R (SEQ ID NO: 139), attL2-F (SEQ ID NO: 140), attL2-R ( Amplified by PCR using SEQ ID NO: 141). The obtained attL1 fragment was digested with restriction enzymes HindIII and attL2 fragment with EcoRI and purified. The PCR reaction conditions were 25 cycles, with a denaturation reaction at 94 ° C. for 1 minute, an annealing reaction at 47 ° C. for 2 minutes, and an extension reaction at 74 ° C. for 1 minute. All subsequent PCR reactions were carried out under the same conditions.

  (2) After cleaving the plasmid pBI221 made by Clontech (Clontech, USA) with restriction enzymes XbaI and SacI, the GUS gene was removed by agarose gel electrophoresis, and the cauliflower mosaic virus 35S promoter (hereinafter referred to as “CaMV35S”). A 35S-Nos plasmid fragment DNA containing a nopaline synthase gene transcription termination region (hereinafter sometimes referred to as “Nos-ter”).

(3) DNA fragments having the sequences of SEQ ID NOS: 142 and 143 below were synthesized, heated at 90 ° C. for 2 minutes, then heated at 60 ° C. for 1 hour, and then allowed to stand at room temperature (25 ° C.) for 2 hours. Annealing was performed to form a double strand. This was ligated to the Xbal-SacI region of the 35S-Nos plasmid fragment DNA to complete the p35S-Nos plasmid. The DNA fragments having the sequences of SEQ ID NOS: 142 and 143 include a BamHI restriction enzyme site at the 5 ′ end, an omega sequence derived from tobacco mosaic virus that enhances translation efficiency, and restriction enzyme sites SmaI, SalI, and SstI in this order.
5'-ctagaggatccacaattaccaacaacaacaaacaacaaacaacattacaattacagatcccgggggtaccgtcgacgagctc-3 '(SEQ ID NO: 142)
5'-cgtcgacggtacccccgggatctgtaattgtaatgttgtttgttgtttgttgttgttggtaattgtggatcct-3 '(SEQ ID NO: 143)

  (4) The p35S-Nos plasmid was digested with the restriction enzyme HindIII, and the attL1 fragment was inserted. This was further digested with EcoRI and the attL2 fragment was inserted to complete the vector p35SG.

<Construction of a vector for construction incorporating a polynucleotide encoding a transcription repressing conversion peptide>
As shown in FIG. 2, p35SSRDXG, a construction vector incorporating a polynucleotide encoding a transcription repressing conversion peptide, was constructed as shown in the following steps (1) to (2).

(1) DNAs encoding the 12 amino acid transcription repressor conversion peptide LDLDLELLRLGFA (SRDX) and designed to have a stop codon TAA at the 3 ′ end, each having the following sequence were synthesized and heated at 70 ° C. for 10 minutes. Thereafter, it was annealed by natural cooling to obtain double-stranded DNA.
5'-gggcttgatctggatctagaactccgtttgggtttcgcttaag-3 '(SEQ ID NO: 144)
5'-tcgacttaagcgaaacccaaacggagttctagatccagatcaagccc-3 '(SEQ ID NO: 145)
(2) p35SG was digested with restriction enzymes SmaI and SalI, and double-stranded DNA encoding the above SRDX was inserted into this region to construct p35SSRDXG.

<Construction of vector for transformation>
As shown in FIG. 3, pBCCK, which is a plant transformation vector having two att sites for recombination with a DNA fragment sandwiched between att sites of the construction vector, is transformed into the following steps (1) to (3 ).

  (1) pBIG (Becker, D. Nucleic Acids Res. 18: 203, 1990) assigned by Michigan State University, USA was digested with restriction enzymes HindIII and EcoRI, and the GUS and Nos regions were removed by electrophoresis.

  (2) Fragment A of Gateway (trademark) vector conversion system purchased from Invitrogen was inserted into the EcoRV site of plasmid pBluescript. This was digested with HindIII-EcoRI and the Fragment A fragment was recovered.

  (3) The recovered Fragment A fragment was ligated with the above pBIG plasmid fragment to construct pBCKH. These can grow only in E. coli DB3.1 (Invitrogen) and are resistant to chloramphenicol and kanamycin.

<Incorporation of MYB106 gene into construction vector>
A gene encoding the transcription factor MYB106 protein derived from Arabidopsis thaliana was incorporated into the construction vector p35SSRDXG as described in the following steps (1) to (3).

(1) From a cDNA library prepared using mRNA prepared from Arabidopsis leaves, a DNA fragment containing only the coding region of Arabidopsis MYB106 gene excluding the stop codon was amplified by PCR using the following primers.
Primer 1 5'-GATGGGCAGATCGCCATGTTGTGATAAGGC-3 '(SEQ ID NO: 136)
Primer 2 5'-GAACATCGTCGCGGAATCGGACGGTGAAGA-3 '(SEQ ID NO: 137)
The cDNA and encoding amino acid sequence of the MYB106 gene are shown in SEQ ID NOs: 2 and 1, respectively.

  (2) As shown in FIG. 2, the obtained DNA fragment of the MYB106 coding region was ligated to the SmaI site of the construction vector p35SSRDXG previously digested with the restriction enzyme SmaI.

  (3) Escherichia coli was transformed with this plasmid, the plasmid was prepared, the nucleotide sequence was determined, and the clone inserted in the forward direction was isolated to obtain a chimeric gene with SRDX.

<Construction of recombinant expression vector>
An expression vector using a plant as a host was constructed by recombining a DNA fragment containing the CaMV35S promoter, chimeric gene, Nos-ter and the like on the construction vector with the plant transformation vector pBCKH. The recombination reaction was performed according to the following steps (1) to (3) using Gateway (registered trademark) LR clone (registered trademark) manufactured by Invitrogen.

  (1) First, p35SSRDXG 1.5 μL (about 300 ng) and pBCKH 4.0 μL (about 600 ng) 5-fold diluted LR buffer 4.0 μL and TE buffer (10 mM TrisCl pH 7.0, 1 mM EDTA) 5.5 μL added.

  (2) 4.0 μL of LR clonease was added to this solution and incubated at 25 ° C. for 60 minutes. Subsequently, 2 μL of proteinase K was added and incubated at 37 ° C. for 10 minutes.

(3) Thereafter, 1-2 μL of this solution was transformed into E. coli (DH5α, etc.) and selected with kanamycin.

<Production of plant transformed with recombinant expression vector>
By the Agrobacterium method, 800 pieces of Torenia leaf pieces were transformed using the MYB106-SRDX gene, and the time of adventitious bud formation and the number of adventitious buds were investigated.

  As shown in the following steps (1) to (3), transformation of torenia is performed with pBCHK-MYB106-SRDX, which is a plasmid in which the DNA fragment containing the chimeric gene is incorporated into pBCCKH, to produce transformed plants. did. Torenia plants are transformed by Aida R, Shibata M (1995) Agrobacterium-mediated transformation of torenia (Torenia fournieri). Breed Sci 45: 71-74; Aida R, Kishimoto, Tanaka Y, Shibata M (2000) Modification of flower color in torenia (Torenia fournieri Lind) by genetic transformation. Plant Sci 153: 33-42.

  (1) First, the obtained plasmid, pBCKH-MYB106-SRDX, was introduced into soil bacteria ((Agrobacterium tumefaciens strain EHA105) by electroporation. The introduced bacteria were antibiotics (kanamycin (Km) 50 μg / mL). Then, the culture was spread on an LB medium plate and placed for about 3 to 5 days at 25 ° C. This was suspended in sterilized water, and a piece of leaf cut into about 3 mm square was soaked. After 1 week, the leaf pieces were transferred to a hygromycin selection medium (Table 3), and then transferred to a new selection medium every two weeks thereafter.

  With the introduction of the MYB106-SRDX gene, adventitious buds started to form on the 40th day, and about 100 shoots were obtained on the 60th day. FIG. 4 is a graph showing the relationship between the number of days (days) after infection (gene transfer) and the number of callus appearance callus.

[Comparative Examples 1-3]
At5g58900-SRDX (Comparative Example 1: corresponding to amino acid sequence of SEQ ID NO: 146, SEQ ID NO: 149), At3g61120-SRDX gene (Comparative Example 2: amino acid of SEQ ID NO: 147, SEQ ID NO: 150) as a control gene instead of MYB106-SRDX gene ), And the At5g03680-ox gene (Comparative Example 3: corresponding to the amino acid sequences of SEQ ID NO: 148 and SEQ ID NO: 151). The time of adventitious bud formation and the number of adventitious buds were compared. The results are shown in FIG.

  As shown in FIG. 4, the gene control gene started to form adventitious buds on the 60th day. In addition, the formation of adventitious buds was 50 or less even after 100 days.

  Thus, as in Example 1, in which the MYB106-SRDX gene was introduced, dedifferentiation or re-differentiation was promoted compared to those in which the control gene of Comparative Examples 1 to 3 was introduced, thereby leading to early and It is thought that many transformed adventitious buds were obtained. That is, it was speculated that the MYB106 gene not added with the SRDX sequence that suppresses the function of the target gene originally has a function of suppressing dedifferentiation or redifferentiation. Moreover, by adding the SRDX sequence to the MYB106 gene and suppressing its function, the efficiency of adventitious bud formation could be greatly improved. By controlling the MYB106 gene, it is highly likely that the efficiency of cultivation and transformation will be greatly improved. By applying it to crops with very low transformation efficiency, such as soybeans and wheat, it is possible to generate great commercial value. There is sex.

Claims (21)

  By producing in the plant a chimeric protein that fuses a transcription factor that promotes transcription of a gene that suppresses dedifferentiation or redifferentiation and a functional peptide that converts any transcription factor into a transcriptional repression factor. A method for producing a plant, characterized by promoting dedifferentiation or redifferentiation of the body. A method for producing a plant according to claim 1,
The plant production method, wherein the transcription factor is the following protein (a ) :
(A) protein having the amino acid sequence shown in SEQ ID NO: 1 A method for producing a plant according to claim 1,
The transcription factor is a protein having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 1 and a function of promoting transcription of a gene that suppresses dedifferentiation or redifferentiation. A plant production method characterized by the above. A method for producing a plant according to claim 1,
The following gene (c) or (d) is used as a gene encoding the transcription factor:
(C) A gene having the base sequence shown in SEQ ID NO: 2 as an open reading frame region (d) Hybridizing under stringent conditions with a gene containing a base sequence complementary to the gene containing the base sequence shown in SEQ ID NO: 2 And a gene encoding a transcription factor that promotes transcription of a gene that suppresses dedifferentiation or redifferentiation A method for producing a plant according to any one of claims 1 to 4,
The method for producing a plant, wherein the functional peptide is a peptide having an amino acid sequence represented by any of the following formulas (1) to (4).
(1) X1-Leu-Asp-Leu-X2-Leu-X3
(Here, in Formula (1), X1 represents 0 to 10 amino acid residues, X2 represents Asn or Glu, and X3 represents at least 6 amino acid residues.)
(2) Y1-Phe-Asp-Leu-Asn-Y2-Y3
(Here, in Formula (2), Y1 represents 0 to 10 amino acid residues, Y2 represents Phe or Ile, and Y3 represents at least 6 amino acid residues.)
(3) Z1-Asp-Leu-Z2-Leu-Arg-Leu-Z3
(Here, in Formula (3), Z1 represents Leu, Asp-Leu or Leu-Asp-Leu, Z2 represents Glu, Gln or Asp, and Z3 represents 0 to 10 amino acid residues.)
(4) Asp-Leu-Z4-Leu-Arg-Leu
(Here, in formula (4), Z4 represents Glu, Gln or Asp.) A method for producing a plant according to any one of claims 1 to 4,
The method for producing a plant, wherein the functional peptide is a peptide having an amino acid sequence represented by any of SEQ ID NOs: 3 to 19. A method for producing a plant according to any one of claims 1 to 4,
The method for producing a plant, wherein the functional peptide is the following peptide (e) or (f):
(E) SEQ ID NO: 20 or a peptide having an amino acid sequence shown in 21 (f) for the amino acid sequence shown in SEQ ID NO: 20 or 21 peptides having at least 90% identity A method for producing a plant according to any one of claims 1 to 4,
The method for producing a plant, wherein the functional peptide is a peptide having an amino acid sequence represented by the following formula (5).
(5) α1-Leu-β1-Leu-γ1-Leu
(In the formula (5), α1 represents Asp, Asn, Glu, Gln, Thr or Ser, β1 represents Asp, Gln, Asn, Arg, Glu, Thr, Ser or His, and γ1 represents , Arg, Gln, Asn, Thr, Ser, His, Lys or Asp.) A method for producing a plant according to any one of claims 1 to 4,
The method for producing a plant, wherein the functional peptide is a peptide having an amino acid sequence represented by any of the following formulas (6) to (8).
(6) α1-Leu-β1-Leu-γ2-Leu
(7) α1-Leu-β2-Leu-Arg-Leu
(8) α2-Leu-β1-Leu-Arg-Leu
(Here, in each formula (6)-(8), α1 represents Asp, Asn, Glu, Gln, Thr or Ser, α2 represents Asn, Glu, Gln, Thr or Ser, and β1 represents Asp, Gln, Asn, Arg, Glu, Thr, Ser or His, β2 represents Asn, Arg, Thr, Ser or His, γ2 represents Gln, Asn, Thr, Ser, His, Lys or Asp Show.) A method for producing a plant according to any one of claims 1 to 4,
The method for producing a plant body, wherein the functional peptide is a peptide having an amino acid sequence represented by any of SEQ ID NOs: 22 to 37, 133, 59, and 60. A method for producing a plant according to any one of claims 1 to 4,
The method for producing a plant, wherein the functional peptide is a peptide having an amino acid sequence represented by SEQ ID NO: 38 or 39. A method for producing a plant according to any one of claims 1 to 11,
A plant production method comprising a transformation step of introducing a recombinant expression vector containing a chimeric gene having a gene encoding the transcription factor and a polynucleotide encoding the functional peptide into a plant cell. . A method for producing a plant according to claim 12,
Furthermore, the production method of the plant body characterized by including the expression vector construction process which constructs | assembles the said recombinant expression vector.   A plant produced by the method for producing a plant according to any one of claims 1 to 13, and modified so as to promote dedifferentiation or redifferentiation.   A chimeric protein comprising a transcription factor that promotes transcription of a gene that suppresses dedifferentiation or redifferentiation, and a functional peptide that converts an arbitrary transcription factor into a transcriptional repression factor.   A chimera comprising a gene encoding a transcription factor that promotes transcription of a gene that suppresses dedifferentiation or redifferentiation, and a polynucleotide encoding a functional peptide that converts any transcription factor into a transcriptional repression factor gene.   A DNA comprising a portion encoding a transcription factor that promotes transcription of a gene that suppresses dedifferentiation or redifferentiation, and a portion encoding a functional peptide that converts any transcription factor into a transcriptional repression factor.   Including a chimeric gene having a gene encoding a transcription factor that promotes transcription of a gene that suppresses dedifferentiation or redifferentiation, and a polynucleotide encoding a functional peptide that converts any transcription factor into a transcriptional repression factor A featured recombinant expression vector.   Recombination including a chimeric gene having a gene encoding a transcription factor that promotes transcription of a gene that suppresses dedifferentiation or redifferentiation, and a polynucleotide encoding a functional peptide that converts any transcription factor into a transcriptional repression factor A transformant comprising an expression vector. The transformant according to claim 19,
A transformant, wherein the transformant is a plant. A kit for producing a plant body,
Recombination including a chimeric gene having a gene encoding a transcription factor that promotes transcription of a gene that suppresses dedifferentiation or redifferentiation, and a polynucleotide encoding a functional peptide that converts any transcription factor into a transcriptional repression factor A kit comprising an expression vector.